Decontamination of laundry exposed to Microsporum canis hairs and spores

Abstract

Objectives

The objective of this study was to determine the efficacy of decontamination of fabric exposed to Microsporum canis hairs and spores by mechanical washing using hot or cold water with or without a sodium hypochlorite additive, and to field test a washing protocol for terry cloth and denim exposed to M canis via direct contact with infected cats.

Methods

Cotton, terry cloth and denim fabric swatches were contaminated with isolated infective spores and hairs and then washed in water at temperatures of 30°C and 60°C, with and without a sodium hypochlorite additive, and with and without mechanical drying. Terry cloth and denim were contaminated by direct contact with infected kittens and washed at 30°C until culture-negative.

Results

All prelaundering samples had >300 colony forming units (cfu)/plate. Experimentally contaminated fabrics were culture-negative, regardless of fabric type, water temperature, the presence or absence of sodium hypochlorite, or tumble drying after one wash. After one wash, 22/34 (65%) of terry cloth towels and 12/20 (60%) denim fabric squares were culture-positive, but the infective load was minimal (1–5 cfu/plate). After two washes in cold water there was no detectable contamination of fabric. The rinse water was not contaminated with spores. The laundry tub was easily decontaminated via mechanical cleaning followed by use of a disinfectant.

Conclusions and relevance

Washable textiles exposed to M canis can be decontaminated via mechanical washes in cold water without the addition of bleach. Two washes are recommended to ensure removal of spores. Laundry can be effectively decontaminated by washing twice in cold water on a long wash cycle (for ⩾14 mins). It is important to ensure maximal agitation (ie, the machine should not be overloaded).

Introduction

Environmental decontamination is an important part of the treatment of cats with Microsporum canis. ¹ The removal of naturally infective material minimizes problems with false-positive fungal cultures and minimizes the risk of disease transmission via fomite contamination. Based on anecdotal clinical experience, recommendations for the decontamination of soft surfaces involve the discarding of disposable items (eg, toys) or laundering.^2,3 Recommendations from human studies are difficult to extrapolate because the primary pathogen is different (Trichophyton species) and target laundry items (ie, socks and underwear) have more direct contact with infection sites than cat bedding. However, studies have shown that mechanical agitation in either hot (60°C) or cold (30°C) water can decrease the fungal load.^4,5

Studies on the decontamination of laundry items exposed to M canis are lacking. The purpose of this study was therefore to determine the effectiveness of home laundry practices for the decontamination of M canis-exposed fabrics. The specific goals were: (1) to determine how easily infective material transfers from exposed to unexposed textiles; (2) to determine the efficacy of decontamination by mechanical washing using hot or cold water with or without a sodium hypochlorite additive; and (3) to field test a washing protocol for terry cloth and denim exposed to M canis via direct contact with infected cats.

Materials and methods

This study was approved by the University of Wisconsin–Madison, School of Veterinary Medicine.

Experimental protocols

Experiment 1: laundry storage and transfer

In this study, four different scenarios were tested to simulate accidental mixing of exposed and unexposed laundry. For each scenario, to simulate accidental mixing of laundry during storage, one 90 mm fabric swatch was contaminated with 100 µl of an isolated infective spore suspension and placed in a biohazard bag (61 × 61 cm) with 20 uncontaminated fabric swatches (90 mm) and tumbled for 1 min in a domestic dryer set to ‘air’. The four scenarios of exposed and unexposed laundry combinations tested were wet to wet, wet to dry, dry to wet and dry to dry. Terry cloth was used for this experiment because towels are the most common laundry items used for animal bedding and drying of cats post-topical therapy. Testing was done in triplicate.

Experiment 2: effects of temperature and sodium hypochlorite

This consisted of several small studies. In the first study, denim, terry cloth and cotton swatches were contaminated with 100 µl of an isolated infective spore suspension and the experiment was conducted using a previously described protocol. ⁶ The swatches were 90 mm in diameter. Twenty sterile fabric swatches were contaminated, allowed to dry and then washed with 10 uncontaminated fabric swatches. The uncontaminated fabric swatches were included to test for transfer during the washing of laundry. In order not to lose the fabric swatches in the laundry tub, each group was placed in a zippered mesh laundry bag (46 × 61 cm). The mesh of the laundry bag was comprised of an open weave of 2 cm² to allow for ready flow of water but not loss of swatches. Laundry experiments were conducted with hot and cold water, each with and without sodium hypochlorite added to the wash water. This was then repeated but with the added step of tumble drying. In this experiment, laundry detergent was not used. Fabric swatches were cultured by pressing both sides of the swatch to the surface of a fungal culture plate three times at the end of treatment; ie, washing or tumble drying. Each sample acted as its own control. The tip of a sterile cotton tip swab was touched to the surface of the contaminated fabric swatch and swabbed onto the surface of a fungal culture plate. Positive growth of M canis documented contamination. Testing was done in triplicate.

Sodium hypochlorite is a common laundry additive used as a disinfectant. In the second experiment, 240 ml of newly purchased 5% sodium hypochlorite (ie, household laundry bleach) was added to the laundry tub and the tub allowed to fill; just prior to putting the laundry in the tub, a 10 ml sample of the water was collected to test for antifungal efficacy. Fungal culture plates were inoculated with 100 µl of the infective spore suspension, allowed to incubate for 6 h and then 100 µl of the collected laundry solution was pipetted and spread on the surface. Water was used as a control. Fungicidal activity was defined as no growth. Testing was done in triplicate. At the end of the last rinse cycle, 240 ml of rinse water was collected and 100 µl was plated onto to a fungal culture plate to test for viable spores. Testing was done in triplicate.

Experiment 3: washing of towels and denim exposed to infected kittens

In this study, terry cloth and denim (30 cm²) was contaminated by gently rubbing a fabric swatch over a kitten infected with M canis. A laundry marker was used to make a circle with a diameter of 50 mm on one side of the fabric, and this was the site of first contact over the most severe lesion. After gently rubbing over the kitten, close inspection revealed hairs trapped in the fibers of the terry cloth and denim. Again, each sample acted as its own control. The surface of the circle was cultured via five swipes of a toothbrush and then inoculated onto a fungal culture plate. Positive growth of M canis documented contamination. Based upon the findings in experiment 1, laundry items were washed in cold water with a generic laundry detergent. Pilot studies using a suspension test determined that the generic laundry detergent had no antifungal properties. In order to simulate an actual laundry load, 2.5 kg ballistic cotton linen was added to each wash load. Contaminated towels (n = 34) were randomly divided into three groups (n = 10, n = 10 and n = 14, respectively). After washing, all of the laundry items were tumble dried and then fungal cultured. Because it was unknown if one wash would be sufficient, towels were laundered and cultured a second time. Twenty denim squares were washed once, air dried and then cultured. A second group of 20 denim squares were washed twice, air dried and then cultured.

Sources of infective material

Samples of isolated plucked hairs or hairs collected from toothbrush combings from lesional, Wood’s lamp-positive untreated cats with naturally occurring infections were used. M canis infection was confirmed via fungal culture and microscopic examination.

Isolated infective spore suspension

Infective spore suspensions were prepared from culture-positive pooled samples as previously described with one variation. ⁷ The infective spore suspension was not filtered, resulting in an infective suspension containing both isolated infective spores and hair fragments; this was considered most representative of infective material shed from cats. Serial dilutions determined were 1 × 10⁵ spores/ml; the spore count was likely higher due to the hair fragments. The suspension was stored at 4°C until used.

Fungal culture procedures

Fungal cultures were grown on BBL Mycosel agar (Becton Dickinson) modified with phenol red as a color indicator. Plates were incubated at 28–30°C for 21 days and examined daily for growth. The final colony-forming unit (cfu)/plate was obtained at day 21; however, the cfu/plate did not change between day 14 and day 21. Confirmation of M canis was via microscopic examination.

Textiles

Denim, cotton and terry cloth were selected for the study because these are commonly encountered textiles in homes, shelters and veterinary clinics.

Contamination protocols

The fabric used in experiment 2 was contaminated by inoculating the surface of the fabric swatch with 100 µl of spore suspension described above.

Detection of contamination postwashing

Fabric was obtained from newly purchased clothing, cut into 90 mm diameter swatches and autoclaved prior to use. Post-test contamination was evaluated by pressing the fabric swatch three times to the surface of a 90 mm fungal culture plate. Replicate Organism Detection and Counting (RODAC) plates (BD Biosciences) designed for environmental studies of microbial contamination were not used in this study because of their small surface size (65 mm). Pilot studies determined that impression testing of fabric was identical to testing with RODAC plates allowing for a larger surface area to be tested (90 mm). Terry cloth towels exposed to infected kittens were cultured using toothbrushes to ensure they were culture-positive before testing. Postwashing, the circle site was cultured by the above impression technique and the remaining surface cultured via toothbrush sampling.

Laundry equipment

Laundry experiments were conducted using a domestic top-loading washing machine and a domestic front-loading dryer. A 14 min agitation cycle was used and fabric was tumble dried for 30 mins. The longest agitation cycle was used (14 mins). High-capacity laundry machines were intentionally not used because the study wanted to mimic a home setting. In order to prevent fomite contamination of test materials, each washing was performed on a separate day. Prior to the study and then after each laundry test day, laundry machines, floors and other surfaces were mechanically cleaned; the surfaces were washed with a generic detergent in water, rinsed with water to remove any residue and then disinfected with a 1:16 dilution of accelerated hydrogen peroxide (Accel Concentrate 4.25%; Virox Technologies).

Environmental cultures

Environmental cultures were obtained using commercial dusting cloths; samples were inoculated by pressing the surface to a fungal culture plate. If fomite contamination was detected, data from that day’s washing were not used. The inside of the laundry tub was cultured after each wash and then decontaminated by wiping the inside of the tub to remove debris and spraying it with 1:16 accelerated hydrogen peroxide. After 10 mins, a water-only wash was performed to ensure no viable spores remained and to remove any disinfectant residue. The inside of the dryer tub and lint trap were cultured, the surface wiped to remove any organic debris and then disinfected with a 1:16 dilution of accelerated hydrogen peroxide. Prior to the study and then on each laundry test day, three cultures were obtained from perceived high-risk areas: the inside of the laundry tub, the dryer tub, the outside of the machines and the floor.

Water temperatures

Laundry experiments were carried out using two temperatures: 60°C (hot water) and 30°C (cold water). The thermostat on the hot water heater was adjusted so that the temperature of the water was 60°C as it entered the washing machine. Using a digital thermometer, the water temperature was monitored while the laundry tub filled with water to determine the water temperature at the start of the wash cycle.

Data analysis

Descriptive data were collected. The number of cfu/plate was counted. For the purposes of this study, good disinfectant efficacy with respect to laundry practices was defined as a decrease in the number of prewashing cfu from >300 cfu/plate to no growth of M canis.

Results

Environmental cultures of the floor and outer surface of the laundry machines revealed consistent disinfection prior to each laundry experiment; no data were discarded owing to fomite M canis contamination. In all washing trials (n = 24), pre- and postenvironmental cultures of the laundry tub were fungal culture-negative.

Experiment 1: laundry storage and transfer

All unexposed fabric swatches were contaminated in all four laundry storage scenarios. Control and test swatches (n = 240) grew >300 cfu/plate. All four scenarios were considered to be of equal risk for the transfer of infective material. The only observational difference was in the wet to wet scenario. Fungal cultures grew more rapidly and produced mature macroconidia within 4–5 days vs 7–10 days.

Experiment 2: effects of temperature and sodium hypochlorite

All prewashed fabric samples were culture-positive (>300 cfu/plate). All of the postwashing samples were culture-negative, regardless of water temperature, the presence or absence of bleach, and with and without tumble drying. Transfer of infective spores from contaminated to uncontaminated swatches during washing was not detected. All water samples containing bleach failed to inhibit the growth of M canis; both control and treated plates grew >300 cfu/plate. M canis was not isolated from any of the final rinse water samples (n = 24). For hot water washes the temperature of the water entering the wash tub was 60°C but was ⩽26°C by the time the wash tub had filled. The inside of the washing tub was not culture-positive at any time.

Experiment 3: washing of towels and denim exposed to infected kittens

A positive fungal culture was found in 22/34 (65%) towel samples, either at the target site (n = 16) or via toothbrush cultures (n = 6) outside of the marked circle. However, the number of cfu/plate ranged from 1–5 cfu/plate (mean 2, median 2). All towels were culture-negative after the second wash. For denim, 12/20 (60%) squares were culture-positive (1–3 cfu/plate; mean 2, median 2) after one wash and all were culture-negative after two washes. Washing in cold water with detergent only was considered to have good efficacy after two washes. The inside of the laundry tub was culture-positive in 2/5 washes (10 and 20 cfu/plate, respectively). In both instances, cat hair was found on the sample clothes. The inside of the dryer tub never cultured positive but the lint trap was culture-positive in the two cases where the laundry tub was culture-positive.

Discussion

Mechanical agitation alone was found to decontaminate fabric exposed to M canis in either hot or cold water, and the addition of a household bleach additive was of no apparent benefit. Prewashing control cultures had >300 cfu/plate and, postwashing, all fabric swatches inoculated with the infective spore suspension were culture-negative. Terry cloth towels and denim directly exposed to infected cats were completely decontaminated after two washes. The culture-positive status of the laundry wash tub and dryer was directly associated with instances where cat hair was present on the surface of the laundry tub. Because decontamination is primarily the result of mechanical agitation, the laundry tub should not be overfilled with clothing. In addition, if cat hair is found on the inside of the laundry tub postwashing, a prudent recommendation would be to wash exposed laundry until cat hair is not found.

Infective material readily transferred from exposed to unexposed laundry when tumbled. This finding was not unexpected and clearly supports the common recommendation to keep exposed and unexposed laundry items separate. Because washers and dryers are in close proximity to each other, accidental contact between exposed unwashed laundry and clean laundry being removed from a mechanical washer is a risk factor. Such exposure could result in fomite contamination of an animal via the bedding and a false-positive fungal culture.

In this study, the temperature of the wash water could be adjusted to 60°C, but it was not possible to maintain this temperature throughout the wash cycle. This is because domestic washing machines do not have the internal mechanisms to heat water that are available in industrial hospital laundries. From a practical perspective, the finding that cold water can be used is important for several reasons. First, from a safety perspective, a hot water setting at 60°C presents a scalding risk. Second, hot water increases energy costs. This is important in veterinary clinics and shelters where large amounts of laundry are washed throughout the day, every day.

It is well established that sodium hypochlorite (household bleach) is antifungal; the most common dilutions for disinfection are 1:32 and 1:100. ⁷ For laundry, the most common label recommendation is to add 120–240 ml to the entire wash tub. The volume of water used per load varies widely in traditional top-loading washing machines and high-efficiency machines from 151–170 l (40–45 gallons) to 53–95 l (14 to 25 gallons), respectively. In order to make a 1:100 dilution of household bleach to fill a washer tub the user would need to add 550–950 ml for high-efficiency machines and 1.5–1.7 l for conventional machines. Not only would the routine use of such volumes be a human health hazard, but it would also be damaging to clothing, plumbing and septic systems if used on a regular basis. In this study, there was no indication that the bleach additive was beneficial. No detectable difference was found between laundry washed with or without bleach. More importantly, there was no evidence of antifungal efficacy in water samples collected when the laundry tub filled. Although laundry detergent itself had no antifungal efficacy against naturally infective material, its use is beneficial because of the surfactant properties that enhance the removal of debris.

It is difficult to compare the results of this study with those in the human literature because the pathogens are different, study design is different and, except for the study by Amichai et al, ⁴ the test inoculum was prepared laboratory cultures of Trichophyton species. Ossowski and Duchmann reported complete decontamination using a domestic washer and a water temperature of 30°C. ⁸ In contrast, Honisch et al and Hammer et al found that 60°C was necessary to decontaminate laundry.^5,6 In the study by Amichai et al, ⁴ socks from patients infected with Trichophyton rubrum were washed at either 40°C or 60°C, and of 81 specimens four samples were positive for T rubrum at the lower temperature and none at 60°C. ⁴ In the study by Hammer et al recommending washing at 60°C, ⁶ it was not reported whether this temperature was maintained throughout the wash cycle. Only one study investigated whether or not fungal organisms were present in the rinse water. In that study, 16% of the fungal load was still present in the rinse water. However, it is important to note that the presence of fungal pathogens was detected using radioactively labeled specimens instead of fungal culture. It is possible that the results reflected the imaging of dead dermatophytes and not viable spores. Of most interest and relevance to the study reported here are the findings by Honisch et al. ⁵ In this study, the impact of wash cycle time, temperature and detergent formulation on domestic laundering was investigated. The study found that the lack of efficacy of laundering at lower temperatures could be compensated for by longer wash times and the use of activated oxygen bleach detergents.

Conclusions

Laundry can be effectively decontaminated by washing twice in cold water on a long wash cycle (⩾14 mins). It is important to ensure maximal agitation (ie, do not overload the machine). Separating exposed laundry from unexposed laundry and routine cleaning of the environment will prevent fomite contamination.